Tissue fusion and cell sorting are processes fundamental to developmental biology with applications in tissue engineering. Tissue fusion, in which two segregated cell populations come together and interact to generate a single tissue, is common throughout development and includes neural tube formation, skeletal formation and myocardial development. After initial contact, cells can create three-dimensional cavities, tubular structures or higher order structures. Disruptions can lead to significant disorders such as spina bifida and cleft palate. For purposes of tissue engineering, it is important to understand and control tissue fusion because strategies are emerging to use cells and aggregates of cells as building units to create larger, more complex three-dimensional tissue structures. For example, tissue fusion is important in organ printing, a process whereby a modified inkjet printer extrudes small volumes of viable cells or cell aggregates along with extracellular matrix proteins (ECMs) to build a three-dimensional structure layer by layer. See Mironov et al., Organ printing: computer-aided jet-based three-dimensional tissue engineering, Trends Biotechnol. 21: 157-61 (2003); Wilson and Boland, Cell and organ printing 1: protein and cell printers, Anat Rec A Discov Mol Cell Evol Biol 272: 491-96 (2003); Bolant et al., Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels, Anat Rec A Discov Mol Cell Evol Biol 272: 497-502 (2003); Jakab et al., Engineering biological structures of prescribed shape using self-assembling multicellular systems, Proc Natl Acad Sci USA 101: 2864-69 (2004). Others are creating microscale modules of cells plus extracellular matrix and assembling these structures to create organoids that can be perfused in vitro. See McGuigan and Sefton, Proc Natl Acad Sci USA 31:11461-114 (2006). Despite its importance, little is understood about the process of tissue fusion and methods are needed to control it. Cell sorting or self-sorting is the ability of two or more cell types to self-organize into distinct regions or layers within a tissue. In development, this process is essential for compartmentalizing cells which leads to neural tube formation, gonad morphogenesis, and development of the heart, lung, and pancreas. Numerous in vitro studies have shown that when two types of mono-dispersed cells are mixed, they will self-assemble a three-dimensional microtissue where one cell type forms the inner core and the other the outer coating of the microtissue. Levels of cell surface adhesion proteins, such as cadherins, influence self-sorting as does cytoskeletal-mediated tension. The ability to control the relative positions of two or more cells within a three-dimensional microtissue has clear applications to tissue engineering. Although much is understood about self-sorting within a single three-dimensional microtissue, there is little understanding of the process when two or more mixed microtissues undergo tissue fusion. Such an event is also important to building larger, more complex tissue structures containing two or more cell types.
A new method for the easy production of large numbers of three-dimensional microtissues (Nap TE and Nap Biotechniqus) has been developed and is disclosed in PCT Patent Publication No. WO 2007/087402 published 2 Aug. 2007 (Application No. PCT/US2007/002050 filed 24 Jan. 2007) (see also, Napolitano et al., Dynamics of the Self-Assembly of Complex Cellular Aggregates on Micromolded Nonadhesive Hydrogels, Tissue Engineering 12: 2087-94 (2007) and Dean et al., Rods, tori, and honeycombs: The directed self-assembly of microtissues with prescribed microscale geometries, FASEB J. 21: 4005-12 (2007)). Briefly, mono-dispersed cells are pipetted onto micro-molded agarose, whereon the cells spontaneously self-assemble three-dimensional microtissues. Microtissue size is controlled by the cell seeding number and mixed microtissues are easily formed by adding a mixture of mono-dispersed cells. Mixed cell suspensions have been seeded into these micro-molded, nonadhesive microgels; the mixed cells segregate into types, with one cell type surrounding the other. Id. Other techniques for aggregating cells are disclosed and reviewed Lin and Chang, Recent advances in three-dimensional multicellular spheroid culture for biomedical research, Biotechnol. J. 3: 1013 (2008).
There is a need in tissue engineering to understand the dynamics and factors governing the fusion of microtissues and the cell sorting that occurs after fusion. The current invention is directed to this aspect of the field.